Abstract Numerous fundamental principles of gene regulation and epigenetics have been unraveled using erythroid cells as a model system. This application will exploit our expertise in erythroid development and chromatin architecture to dissect the diverse activities of CTCF, a central multi-functional transcription factor. CTCF can activate or repress transcription, slow elongation by RNA polymerase 2, form long range chromosomal contacts, block enhancer activity, and form chromatin domain boundaries. CTCF bound sites (CBS) can be tissue-specific or tissue-invariant. A leading question in the field is how does CTCF exert these distinct activities. Other key open questions are how lineage-dependent functions and chromatin binding by CTCF are specified. Utilizing an auxin-inducible CTCF degradation system in an erythroid cell line we found that, unexpectedly, CBS are turned over with highly variable kinetics. This leads to the hypothesis that location- specific differences in susceptibility to degradation may be a reflection of CTCF chromatin residence time that is locally specified by chromatin context. We further hypothesize that such differences in residence times are critical features of distinct CTCF mechanisms of action. In Specific Aim 1 we will first determine erythroid- and maturation stage-specific CBS genome wide, globally assess auxin-modulated CBS dynamics in both immature and mature erythroid cells, and examine consequences of acute CTCF depletion on gene expression. The determinants of CBS specificity and dynamics will be dissected via genome editing and targeted proteomics (ChIP-SICAP). In Specific Aim 2, we will test the hypothesis that CBS dynamics are a function of chromatin residence time. This will be measured by single molecule tracking (SMT) of CTCF, to be carried out in collaboration with Dr. Melike Lakadamyali at UPenn. These studies will include perturbative manipulations of CTCF. By combining acute CTCF degradation with PRO-seq we discovered that many genes with CBS near the start site exhibit increased transcription in the anti-sense direction upon CTCF loss. This suggests a novel role of CTCF in governing transcription directionality. In Specific Aim 3 we will determine the mechanism by which CTCF regulates transcription directionality, exploring chromatin looping versus functioning as an elongation barrier as possible alternative mechanisms. Single molecule RNA FISH will be employed to study the dynamics and mechanisms of transcription directionality. These studies address longstanding questions as to how CTCF accomplishes its diverse functions, including new ones discovered here, and are expected to have broad ramifications for the fields of gene regulation and nuclear architecture.